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Five gallbladder cancer (GBC) cell lines were examined for morphological changes in collagen gel culture. GBh3 and HUCCT-1 cells formed tubules in response to treatment with epithelial growth factor (EGF) and hepatocyte growth factor (HGF), and showed high levels of expression of E-cadherin (ECD), and low levels of SNAIL, vimentin, transforming growth factor (TGF)-β, and nucleostemin (NS). In contrast, the GBd15 and FU-GBC-1 cell lines treated with EGF and HGF showed a scattering phenotype, and expressed low levels of ECD and high levels of SNAIL, vimentin, TGF-β, and NS. All cell lines expressed the EGF receptor, c-Met, EGF, and TGF-α, but not HGF. Transforming growth factor-β was upregulated by EGF. Knockdown of the EGF receptor abrogated both tubule formation and scattering, whereas KD of TGF-β abrogated only scattering. Knockdown of EGF induced nuclear translocation of β-catenin and Wnt-related NS induction in the scattering cell lines, but not in the tubule-forming cell lines, whereas KD of glycogen synthase kinase-3β in the tubule-forming cell lines resulted in the nuclear translocation of β-catenin and Wnt-related NS induction in response to EGF treatment. These results suggest that EGF enhances epithelial–mesenchymal transformation and acquisition of stemness in GBC cells with a scattering phenotype through the activity of β-catenin. Repression of ECD in scattering GBC cells induced the release of β-catenin from the cell adhesion complexes along the plasma membrane and its translocation to the nucleus to activate Wnt signaling, which upregulated NS. (Cancer Sci 2012; 103: 1165–1171)
Biliary tract cancer is the sixth leading cause (5.1%) of cancer death in Japan, with 17 000 BTC patients dying in 2009. Biliary tract cancer shows poor prognosis in spite of aggressive treatment, and the 5-year survival is only 18%. Nodal metastasis is the most significant prognostic factor for GBC.[2-4]
Epithelial growth factor receptor is a key factor in epithelial malignancies, and its activity enhances tumor growth, invasion, and metastasis. Biliary tract cancers express EGFR in 60.7% of cases. The EGFR-overexpressing GBC cases show poorly differentiated histology and decreased survival of 1.5 years in median survival. Amplification and point mutations of the EGFR gene have been reported to be 1% and 15–26.5%, respectively, in GBC.[8-10] The HGF receptor c-Met is involved in the early carcinogenesis of BTC. c-Met is expressed in 74% of invasive GBC and is associated with invasive depth. Because HGF is secreted from fibroblasts, c-Met activation depends on the cancer–host interaction. Transforming growth factor-β is widely expressed in advanced GBC and is associated with angiogenesis and tumor-associated macrophage infiltration as well as with stromal fibrosis.[14, 15]
Epidermal growth factor receptor, c-Met, and TGF-β have recently been implicated in the process of EMT.[16-19] Epithelial–mesenchymal transition comprises a switch in cell differentiation from polarized epithelial cells to contractile and motile mesenchymal cells. In EMT-type cells, the reduction of the epithelial marker ECD occurs in parallel with the induction of the mesenchymal marker VIM. Epithelial–mesenchymal transition occurs during cancer progression and enhances invasion and metastasis.
The present study aimed to clarify the relationship between morphogenesis, EMT, and growth factors in GBC cells.
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In the present study, we examined the effect of growth factors on morphogenesis and scattering of GBC cells. The scattering phenotype of GBd15 and FU-GBC-1 cells was enhanced by treatment with EGF and HGF. Scattering cancer cells show spindle fibroblastic shape and poor cell-to-cell attachment. In the present study, these cell lines showed decreased ECD and SNAIL expression, which is a key phenotype of EMT. They also expressed TGF-β at high levels; TGF-β is closely associated with EMT induction. Moreover, the scattering cell lines expressed NS at high levels. Nucleostemin is one of the stemness markers, and high NS expression is considered to be related to the acquisition of the stem cell phenotype. This pattern of expression is compatible with EMT of cancer cells.
β-Catenin is an ECD-bound cytoplasmic molecule with a link to the cytoskeleton. The dissociation of β-catenin from ECD results in its translocation into the nucleus to act as a transcription factor in Wnt activation. Mutations in the APC gene cause inactivation of the ECD complex and nuclear translocation of β-catenin, which is important for carcinogenesis. Thus, alterations in the ECD and β-catenin complex affect multiple pathways to enhance the malignant activity of cancer cells.
The present data showed that the EGF-induced β-catenin translocation into the nucleus occurs in the GBC cells with a scattering phenotype, and is associated with Wnt-related increase in NS. In contrast, in GBC cells with a tubule-forming phenotype, nuclear localization of β-catenin was not detected in the presence or absence of EGF. However, KD of GSK-3β resulted in nuclear translocation of β-catenin and Wnt-related NS induction. Downregulation of ECD, therefore, induces nuclear translocation of β-catenin, which subsequently increases stemness by Wnt activation. β-Catenin, therefore, plays an important role in the acquisition of stemness in the scattering GBC cells, in which ECD is repressed, inducing the release of β-catenin from the cell adhesion complex on the cytoplasmic membrane. The free β-catenin translocates to the nucleus to activate Wnt signaling, which upregulates NS. In tubule-forming GBC cells, the forced release of β-catenin from the cell adhesion complex by GSK KD caused a scattering phenotype and NS upregulation. In clinical studies, nuclear and/or cytoplasmic translocation of β-catenin was found in half of the GBC cases and was associated with poorly differentiated histology.
Activation of Wnt signaling by β-catenin nuclear translocation affects the behavior of mesenchymal stem cells and also mediates EMT. Moreover, EMT is linked to the acquisition of the stem cell phenotype of cancer cells. Our current data is compatible with this mechanism. Gallbladder cancer cells with a scattering phenotype are thought to possess an EMT phenotype with high cancer stem cell competence, which is related to more aggressive progression and metastasis of the disease. Moreover, EMT-type cells with stemness are responsible for drug resistance.[27, 28]
In our study, the EMT phenotype was closely associated with ECD expression. Gallbladder cancer cells with low ECD expression showed a scattering phenotype in response to EGFR activation, whereas GBC cells with high ECD expression showed a tubule-forming phenotype. Reduced ECD expression is found in more than 60% of GBC cases.[29, 30] Downregulation of ECD is more pronounced in advanced GBC cases than in early cases, and is associated with poorer prognosis. Repression of ECD is caused by promoter DNA methylation, gene mutation, and transcriptional regulation. Gene silencing of ECD by methylation of the promoter is detected in 41% of BTC cases. High frequency of loss of heterozygosity of ECD is found in GBC. In the negative regulation of ECD, certain transcription factors have been identified as playing a role, including ZEB-1, ZEB-2, Twist, Slug, and SNAIL. These factors are observed during the EMT of cancer cells. In the present study, SNAIL expression was inversely correlated with ECD expression. Expressions of Twist, ZEB-1, and Slug were also increased by EGF treatment in association with ECD repression in GBd15 cells. In contrast, HUCCT-1 cells showed the same level of SNAIL expression as GBh3 cells, whereas ECD expression in HUCCT-1 cells was lower than that in GBh3 cells. Methylation of the promoter of the ECD gene was detected in HUCCT-1 cells (data not shown). Recent studies identified specific microRNAs associated with ECD repression or EMT. A candidate oncogenic miRNA, miR-21, was found to induce TGF-β-related EMT, and miR-200 is downregulated in cancer cells, which results in ZEB-1/-2-related EMT. These factors should be examined in GBC.
Our data showed the significance of EGFR in the induction of EMT in GBC cells. Activation of EGFR inactivates GSK-3β and upregulates SNAIL, which results in ECD repression and EMT in uterine cervical cancer. Epidermal growth factor receptor also enhances ubiquitination and degradation of ECD by CDC42 activation through the Src pathway. In contrast, in cells expressing ECD and showing stable cell-to-cell attachment, the activity of the EGFR–ERK pathway is decreased and β-catenin–T lymphocyte-specific transcription factor (TCF) signaling is inhibited. In dense culture of ECD expressing airway epithelial cells, EGFR activation promotes cell differentiation with mucin production. E-cadherin regulates EGFR by promoting the formation of a complex with the extracellular domain of ECD. The intrinsic tyrosine kinase activity and dimerization of the EGFR in cells grown in sparse culture are induced by EGF, whereas these activities are not induced in cells grown in dense culture. Thus, interaction between EGFR and ECD generates both tubulogenesis and scattering. Decreased ECD levels enhance EGFR-related ECD downregulation in a vicious cycle, which results in the inactivation of GSK-3β, β-catenin–Wnt activation, increment of stemness, and EMT.
In cholangiocellular carcinoma cell lines, the anti-EGFR antibody cetuximab is partially effective in EGFR-expressing cells. KRAS mutations affect the efficacy of cetuximab in these cells. Gefinitib, a selective EGFR tyrosine kinase inhibitor, inhibits the phosphorylation of EGFR, ERK, and AKT, and induces G1 arrest and apoptosis by upregulating p21 and p27, and BAX activation in GBC cells. Epidermal growth factor receptor targeting is, therefore, critical in the treatment of GBC.